The research objectives of this proposal are several and are based on very promising initial results. These include: (a) To synthesize a series of inhibitors of key enzymes that metabolize glutomate, aspartate, and ammonia, namely glutamine synthetase, asparagine synthetase, and aspartokinase. These compounds are selected for their resemblence to tightly bound intermediates (e.g., gamma-Glu-P, Asp-AMP, or Asp-P) or transition states of the enzyme-catalyzed reactions. (b) To study and apply these inhibitors as potential cancer chemotherapeutic agents, as probes of catalytic mechanism, and as affinity ligands for chromatography and active-site labeling. (c) To develop more fully the theoretical basis for defining exactly the various modes of action of enzyme regulators (inhibitors or activators) through the use of equilibrium isotope exchange techniques. This approach may prove to be more insightful than initial velocity probes in many cases, and could, through computer-assisted models now available, be extended to pathway-level investigations. (d) To study the multiple forms of gluatmine synthetase isolated from the extreme thermophile, Bacillus caldolyticus, in terms of regulation, structure, and function.